Fractionation of plasma using glycine and polyethylene glycol

ABSTRACT

CONCENTRATES OF ANTIHEMIPHILIC FACTOR A AND PROTHROMBIN COMPLEX ARE PREPARED FROM CITRATED BLOOD PLASMA BY AN INITIAL FRACTIONATION WITH GLYCINE FOLLOWED BY MULTIPLE FRACTIONATIONS OF THE AHF-CONTAINING PRECIPITATE AND THE PROTROMBIN COMPLEX-CONTAINING SUPERNATE WITH POLYETHYLENE GLYCOL, THE AHF-CONTAINING FRACTION BEING GIVEN AN ADDITIONAL FRACTIONATION WITH GLYCINE AND THE PROTHROMBIN COMPLEX-CONTAINING FRACTION BEING GIVEN AN INTERMEDIATE ADSORPTION WITH TRIBASIC CALCIUM PHOSPHATE.

Aug. 8, 1972 L. F. FEKETE ETA]. 3,682,881

FRACTIONATION 0F PLASMA USING GLYCINE AND POLYETHYLENE GLYCOL Filed Oct. 2, 1970 FROZEN 0R FRESH CITRATED PLASMA (L8M GLYCINE) PLASMA SUPERNATANT AHF PRECIPTATE DILUTION WITH 0.9% NaCl 3 0/ PEG TO 30% PEG SALTS PLASMAPROTEIN FIBRINOGEN PPT pp IO% PEG DILUTION WITH 0.9% NaCl TO ORIGINALVOLUME SUPT. AHF PRECIPITATE 0.5% TRICALCIUM PHOSPHATE I LBMGLYCINE PROTHROMBIN UNABSORBED PROTEIN COMPLEX PPT SUPT. AHF CONCENTRATE 0.! M NO CITRATE ELUATE TRICALCIUM PHOSPHATE 5% PEG 20% PEG TRICALCIUM PHOSPHATE SALTS PROTHROMBIN COMPLEX INVENTORS EDWARD SHANBROM LAJOS F. FEKETE BY/ivgd/yw ATTORNEY United States Patent US. Cl. 260-112 B 7 Claims ABSTRACT OF THE DISCLOSURE Concentrates of antihemophilic factor A and prothrombin complex are prepared from citrated blood plasma by an initial fractionation with glycine followed by multiple fractionations of the AHF-containing precipitate and the prothrombin complex-containing supernate with polyethylene glycol, the AHF-containing fraction being given an additional fractionation with glycine and the prothrombin complex-containing fraction being given an in termediate adsorption with tribasic calcium phosphate.

CROSS-REFERENCES TO RELATED APPLCATIONS This is a continuation-in-part of co-pending application Ser. No. 679,240, filed Oct. 30, 1967 now abandoned and co-pending application Ser. No. 834,883, filed June 19, 1969 now US. Pat. 3,560,475. Application Ser. No. 679,240 is a continuation-in-part of application Ser. No. 634,839, filed May 1, 1967 now abandoned, and has been refiled as a continuation application May 1, 1970.

This invention relates to a method for the purification of blood coagulation components and, more particularly, to the preparation of concentrates of antihemophilic factor A and prothrombin complex from citrated blood plasma.

The process of blood coagulation is a complicated physiological activity and involves the interaction of numerous substances found in normal whole blood. It is know that certain factors associated with the blood coagulation mechanism are absent or seriously deficient in certain individuals. In those patients suifering from classical hemophilia, antihemophilic factor A (AHF, Factor VIII) is deficient. In those patients afllicted with hemophilia B, plasma thromboplastin component (PTC, Factor IX) is missing from the blood.

In the development of modern blood banking programs involving the collection and storage of large quantities of blood and blood components, the establishment of adequate preservation systems is critical. Since World War II it has been common practice to collect blood in a solution of citric acid, sodium citrate and dextrose, known as ACD blood. The problem of preserving blood is much simplified, however, when it is reduced to preservation of various blood components since it is easier to meet the environmental requirements of the separate components than of whole blood.

Moreover, it is wasteful and even detrimental to the patient to administer more blood components than required. Thus, the hemophiliac needing certain blood coagulation factors ideally is given only those factors required or at least a purified concentrate of those factors.

For the treatment of classical hemophilia, antihemophilic factor A fractions, 20 to 35 times purified in terms of activity per miligram of protein and 10 times concentrated, have been prepared by glycine-precipitation of either fresh or frozen plasma by Webster et al., Amer. J.

"ice

Med. Sci. 250, pp. 643-651 (1965) or by cryoprecipitation of fresh-frozen plasma, Pool, New Eng. J. Med. 273, pp. 1443-47 (1965) and Hershgold et al., J.'Lab. Clin. Med. 67, pp. 23-32 (1966). While there is no doubt that these regular glycine-precipitated AHF concentrates represent a significant therapeutic advance in the treatment of hemophilia A, they do not represent the ideal therapeutic agent.

A major disadvantage in the use of plasma cryoprecipitate fractions as starting material for the preparation of antihemophilic factor A is that in practice only about 30% yield of AHF is obtained whereas yields of AHF on the order of about 70% are obtained by the glycine-precipitation of whole plasma. However, when citrated plasma is used for the AHF fractionation, the AHF- depleted plasma generally is unsuitable for preparation of plasma thromboplastin component (PTC, Factor IX) and other coagulation factors of the prothrombin complex. That is, citrated plasma is known to inhibit the adsorption of anticoagulation factors on calcium phosphate, as shown by Steinbuch and Solier, Propl. Geriatol Pereliv. Krovi. 11, pp. 15-21 (1966) (see Chem. Abstracts 66, p. 9226 (1967)).

Consequently, a method by which citrated plasma could be used directly for the prepartion of separate concentrates of antihemophilic factor A and prothrombin complex would find much use in the field of blood coagulation therapy.

Accordingly, it is an object of this invention to provide a method for the purification of blood coagulation components.

It is another object of this invention to provide a method for the preparation of concentrates of AHF and prothrombin complex from citrated blood plasma.

Other objects and advantages will be apparent to those skilled in the art after reading the disclosure herein.

In accordance with the present invention, fresh or frozen citrated plasma is mixed with glycine, using a final glycine concentration of from about 1.3 to about 1.8 molar, to produce an AHF-containing precipitate and a prothrombin complex-containing plasma supernatant. Each of the respective precipitate and supernate fractions are then subjected to multiple fractionations with polyethylene glycol, the AHF-containing fraction being given an additional precipitation with glycine and the prothrombin complex-containing faction being given an intermediate adsorption with tribasic calcium phosphate.

While the specification concludes with claims particularly pointing out and distinctly claiming the subject matter regarded as forming the present invention, it is believed that the invention will be better understood from the following detailed description taken in connection with the accompanying drawing which illustrates a preferred embodiment of the invention. In the drawing, FIG. 1 is a schematic chart of a preferred embodiment of the method of preparing the separate concentrates of antihemophilic factor A and prothrombin complex from citrated blood plasma.

As used herein, the term prothrombin complex refers to a concentrate of blood proteins which are active in the coagulation process comprising principally prothrombin (Factor II), proconvertin (Factor VII), antihemophilic factor B (Factor IX) and Stuart-Prower factor (Factor X).

After separation of the AHF containing fraction from the prothrombin complex-containing fraction by the glycine precipitation step, each of the respective fractions is separately subjected to several polyethylene glycol precipitation steps. The polyethylene glycol used as a precipitating agent in this inventtion is a high molecular weight polymer which is generally produced by reacting ethylene oxide with ethylene glycol or water and has the following structure:

in which n represents the average number of oxyethylene groups. According to the present invention the polyethylene glycol should be non-toxic and can range in molecular weight from about 200 to about 20,000. It preferably has a molecular weight of from about 440 to about 6,000. PEG 4,000, which is a polyethylene glycol product having an average molecular weight of about 4,000, i the preferred product of this group. The precipitation with these polyethylene glycol polymers is preferably conducted at normal room temperature (about 25 C.).

In the preferred method of the invention for obtaining the prothrombin complex, the plasma supernatant from the glycine precipitation step is suspended in normal physiological saline (about 0.9% NaCl), preferably to a dilution of about 1:1, the pH is adjusted to from about 6.0 to about 7.2, and the resulting suspension is precipitated with polyethylene glycol to a final concentration of about 30% PEG. The resulting plasma protein precipitate is then separated from the supernatant. The supernatant, which contains the undesired citrate and glycine salts, is discarded. The plasma protein precipitate is resuspended in normal physiological saline, the pH is ad justed to from about 6.8 to about 7.2, and tribasic calcium phosphate is thoroughly mixed with the suspension to adsorb the coagulation factors. The resultant tribasic calcium phosphate adsorbed-protein precipitate is then thoroughly mixed with from about 0.05 M to about 0.2 M trisodium citrate followed by recovery of the resulting supernatant which contains the desired coagulation factors. The supernatant is subjected to a succession of two polyethylene glycol precipitations, first at a pH of from about 6.8 to abut 8.0 and to a final concentration of from about to about polyethylene glycol with retention of the resulting supernatant, and then at a pH of from about 5.0 to about 5.4 and to a final concentration of at least about 20% polyethylene glycol by weight of said retained supernatant with retention of the resulting precipitate. The precipitate, which contains the active prothrombin complex, is then preferably suspended in citrated saline to a final volume of from about one twentyfifth to about one tenth the volume of the suspension of the starting blood plasma.

After suspension of the plasma protein precipitate has been achieved in the foregoing method of the present invention, the adsorption of the coagulation factors is carried out by adjusting the pH of the suspension to within a range of from about 6.8 to about 7.2 followed by adding a small amount of tribasic calcium phosphate. The tribasic calcium phosphate used in this invention is a polymeric type material which can be described by the formula 10CaO-3P O -H O and, alternatively, by the formula Ca (OH) (PO The use of from about 0.5% to about 2% by weight of tribasic calcium phosphate has been found to be suitable for the adsorption of the coagulation factors and a concentration of about 1% is preferred. The tribasic calcium phosphate preferably is allowed to mix with the suspension for about to about 30 minutes in order to provide for substantially maximum adsorption of the coagulation factors.

The tribasic calcium phosphate adsorbed-protein precipitate is separated by centrifugation and then suspended in trisodium citrate, preferably to a volume of from about one tenth to about one two hundredth the volume of the starting blood plasma. An aqueous solution of up to about 0.005 M trisodium citrate can optionally be used for the elution of undesirable contaminating proteins, when present, whereas from about 0.05 M to about 0.2 M trisodium citrate is used for elution of the coagulation factors from the tribasic calcium phosphate adsorbant. The precipitate preferably is suspended in the trisodium citrate with constant stirring for about 15 to about 30 minutes in order to provide for substantially maximum elution of the respective contaminating proteins and the desired coagulation factors. Separation of these substances from the tricalcium phosphate particles is preferably carried out by centrifugation accompanied with constant stirring.

After carrying out the two successive polyethylene glycol precipitations, the resuspended polyethylene glycol precipitate, which contains the active prothrombin complex, is preferably lyophilized or freeze-dried after the addition of an anticoagulant, for example, heparin, in an amount of from about one to about ten units per mil. (U.S. Pharmacopoeia units), adjustment of the pH to about 6.8, and filtering to remove any undesired particles and at the same time to obtain a sterile product without heating. The dry, lyophilized product is stable and can be reconstituted with water prior to use for intravenous, subcutaneous or intramuscular administration. Intravenous, subcutaneous or intramuscular administration of the prothrombin complex is useful to correct temporarily Factors II, VII, IX and X in persons deficient in such factors, to stop bleeding episodes, and/or prevent expected bleeding episodes, for example, bleeding disorders associated with liver disease or hemorrhagic diseases of the newborn.

In the above procedure, the preferred alkaline reagent for pH adjustment is an aqueous solution of about 1 N sodium hydroxide. Other conventional alkaline reagents, for example, sodium bicarbonate, can be used in place of sodium hydroxide. The preferred acid reagent for pH adjustment, when required, is an aqueous solution of about 1 N hydrochloric acid. Other conventional acidifying reagents, for example, acetic acid, can be used in place of hydrochloric acid.

In the preferred method of the present invention for obtaining the concentrate of antihemophilic factor A, the AHF-containing precipitate from the glycine precipitation step is fractionated by two successive precipitations with polyethylene glycol, first at a pH of from about 6 to about 7 and to a final concentration of from about 3% to about 4% polyethylene glycol by weight of the starting material followed by recovery of the resulting supernate, second at a pH of from about 6.4 to about 7.4 and to a final concentration of about 10% polyethylene glycol by weight of the supernate followed by the recovery of the resulting precipitate, and then fractionated by precipitation of said recovered precipitate with an aqueous glycine solution having a molarity of about 1.8.

In the polyethylene glycol precipitation step, the AHE- containing precipitate is first redissolved and then the redissolved material is subjected to the above-described two successive precipitations with polyethylene glycol, followed by recovery and redissolution of the second precipitate. The redissolved polyethylene glycol precipitated fraction is then subjected to precipitation with glycine.

Recovery of the polyethylene glycol and glycine precipitated fractions for use in this invention can be accomplished, for example, by centrifugation or filtration of the respective precipitates or by similar such procedures. Redissolution of the recovered precipitates can be achieved by warming and agitating in citrated saline solution. In the case of the redissolution of the AHF- containing starting precipitate, it is preferred to use a glycine citrated saline solution and to increase the volume of the mixture to about one twentieth the volume of the original plasma which the starting precipitate represents. The polyethylene glycoland glycine-precipitated fractions are preferably redissolved with citrated saline solution to increase the volume of the fractions to about one two-hundredth the volume of plasma which the respective precipitated fractions represent.

It is also preferred to purify each of the respective redissolved starting precipitate and polyethylene glycoland glycine-precipitated fractions by clarifying with additional centrifugation and/or filtration to remove any insoluble matter.

The above fractionation by successive precipitation with polyethylene glycol and glycine has been found to provide a highly soluble AHF concentrate of high potency which can be frozen and rendered stable, such as by lyophilization, and retained under ordinary refrigeration conditions for periods of a year or longer. The potency of each batch of material prepared by the above fractionation method can be precisely determined so that the administering physician can know exactly how much AHF his patient receives.

Since the redissolved AHF concentrate prepared by the above fractionation method has more than ten times the AHF activity of an equal volume of plasma, the hemophiliac can be given a quantity of AHF which the heart could not otherwise tolerate. Even more importantly, the AHF activity in the above-prepared concentrate is contained in less than 2.5% the amount of protein present in plasma providing an equal amount of AHF activity. This lower protein content minimizes the likelihood of allergic reactions by the hemophiliac recipient and reduces the possibility of overloading the circulatory system.

The concentrate of AHF which has been prepared by successive precipitation with polyethylene glycol and glycine can be further purified by treatment with ECTEOLA cellulose resin. This purification can be carried out either before or after the polyethylene glycol and glycine precipitation and may be done by column or batch techniques. The concentrate purified by this method has the additional advantage in that it can also be administered intramuscularly as well as by intravenous administration methods generally used in the case of the AHF concentrate which has not been treated with the ECTEOLA cellulose resin. The AHF concentrate purified with ECTEOLA cellulose resin has been found to be free of fibrinogen by the addition of thrombin and by immunoelectrophoresis.

IAS used herein, the term ECTEOLA cellulose resin refers to a modified cellulose 'which contains active basic substituents introduced into the cellulose molecule by reaction with epichlorohydrin and triethanolamine. Methods of preparation of ECTEOLA cellulose resins are described in general by Sober and Peterson, J. Am. Chem. Soc. vol. 76, pp. 1711-12 (1956); id., vol. 78, pp. 751- 55 (1956); vol. 78, pp. 756-63 (1956); and Peterson and Sober, Biochem Preparations, vol. 8, pp. 43-4 (1961).

ECTEOLA cellulose resins are available commercially. However, it has been found desirable to initially treat these resins by recycling them with caustic soda before use in the herein-defined purification procedure.

In the purification procedure with ECTEOLA cellulose resin, the resin preferably is first equilibrated with a chloride buffer solution having a concentration of about 0.8% NaCl and then poured into a chromatographic glass column. The AHF concentrate which is desired to be purified is then applied to the column and finally eluted with a chloride buffer solution having a molarity of about 0.5.

Other methods of further purifying the AHF concentrate of the present invention will be apparent to those skilled in the art after reading the invention described herein.

The following examples further illustrate the present invention although the invention is not limited to these specific examples which are provided for purposes of illustration and not limitation. All parts and percentages herein are on a weight basis unless otherwise specified.

EXAMPLE I Fresh citrated plasma (from ACD preserved blood) is mixed with an aqueous solution of glycine to a concentration of 1.8 molar glycine and the resulting suspension is centrifuged. The precipitate, which contains a concentrate of AHF, is retained for subsequent treatment in Examples 4 and 5, below. The plasma supernatant (pH 6 7) is diluted with an equal volume of normal 6 physiological saline and then mixed with polyethylene glycol having a molecular weight of about 4000 (PEG 4000) to a concentration of 30% PEG. The resulting plasma protein participate is separated from the super-' natant by centrifugation and the supernatant, which contains the undesired citrate and glycine salts, is discarded.

The plasma protein precipitate is suspended in normal physiological saline to a concentration of 10% (weight/ volume) and the pH adjusted to 7.2 with 1 N NaOH. 500 grams of tribasic calcium phosphate is then added to 50 liters of the plasma protein suspension and the mixture stirred for about 30 minutes. The suspension is then centrifuged and the supernatant discarded. The retained precipitate is suspended in 0.1 M trisodium citrate to a final volume of 5 liters. The suspension is again centrifuged and the precipitate discarded. The pH of the retained supernatant (about 5 liters) is then adjusted to 7.2 with l N HCl, polyethylene glycol 4000 is added to a final concentration of 5%, and the suspension stirred for about 30 minutes. The suspension is clarified by centrifugation, with retention of the supernatant and discardal of the precipitate. The pH of the retained supernatant is then adjusted to 5.2 with 1 N HCl, and polyethylene glycol 4000 is added to a final concentration of 20%. The suspension is centrifuged and the precipitate that is recovered is dissolved in citrated saline (1 part 0.1 M trisodium citrate to 4 parts 0.9% sodium chloride) to a final volume of 2 to 5 liters, which is equivalent to one twenty fifth to one tenth the volume of the original plasma protein suspension. Heparin is added in an amount of one unit per ml., and the solution is clarified and sterilized by passage through a combination of graded pore sizes of membrane filters. The solution is filled under aseptic conditions in 10 to 30 ml. sterile bottles, freeze dried and capped with stoppers. The freeze-dried material can be reconstituted with sterile water and then administered intravenously, subcutaneously or intramuscularly to patients who are deficient in one or more of the abovementioned coagulation factors, particularly Factor IX. The Factor IX activity of the reconstituted product is about 20 times as great as an equal volume of normal whole plasma and is contained in about one eighteenth the amount of protein in normal whole plasma.

EXAMPLE 2 Example 1 is repeated up to the point of suspending the plasma protein precipitate in normal physiological saline. Tribasic calcium phosphate N.F. is added to the suspension to a concentration of 1%. The resultant tribasic calcium phosphate precipitate is then suspended in 0.005 M trisodium citrate to a volume equal to one two-hundredth the original plasma volume. After mixing for an interval of thirty minutes at 5 C., the suspension is centrifuged to remove undesired contaminating materials in the resulting supernatant. The retained precipitate is then suspended in 0.2 M trisodium citrate to a volume equal to one two-hundredth the original plasma volume. After mixing for an interval of thirty minutes at 5 C., the suspension is centrifuged to remove the tribasic calcium phosphate particles. The pH is adjusted to 6.8 with 2 N acetic acid and PEG 4000 is added to provide a final concentration of 10%. The precipitate that forms is discarded. The supernatant is then adjusted to pH 5.2 and sufiicient PEG 4000 is added to provide a final concentration of 20%. The suspension is centrifuged and the precipitate that is recovered is dissolved in citrated saline (one part of 0.1 M sodium citrate to 9 parts of a 5% sodium chloride solution) to a volume to provide 40 units of Factor IX per ml. The pH is adjusted to 6.8 with 1 N sodium hydroxide. Heparin in an amount of 3 units per ml. is added, and the solution is filtered through a series or combination of graded pore sizes of Millipore filters. The solution is filled under aseptic conditions into sterile ml. glass bottles in units of 20 ml. of solution per bottle. After shell-freezing and drying from the frozen state under aseptic conditions, the bottles are closed with sterile stoppers under vacuum and capped. The dry product prepared in accordance with this example can be used for intravenous or intramuscular injection after reconstitution with 20 ml. of sterile water per each unit of dry product.

EXAMPLE 3 Example 2 is repeated except that frozen plasma is used instead of fresh plasma. The plasma is obtained from a plasma pool ranging in age from two weeks to two months and stored at -25 C. for various periods of time ranging up to nine weeks before processing in accordance with the procedure of Example 2. The prothrombin complex prepared in this manner is assayed after reconstitution with sterile water as follows:

(a) Factor II content: The solution is assayed for prothrombin activity by the methods of Ware and Seegers, Am. J. Clin. PathoL, vol. 19, pp. 471-82 (1949) and Wagner et al., Blood Coagulation, Hemorrhage and Thrombosis, edited by Tocantins and Kazal, published by Grune and Stratton, New York, pp. 159-165 (1964).

(b) Factor IX content: The solution is assayed for PTC activity according to the kaolin-activated partial theromboplastin time correction method described in the Hyland Reference Manual of Coagulation Procedures, published by Hyland Laboratories, Los Angeles, Calif., pp. 19-21 (2d ed. 1964).

(c) Thrombin activity: The presence of thrombin is tested for by determining the clotting time of recalcified normal plasma at various dilutions according to the procedure of Bidwell and Dike, Treatment of Hemophilia and Other Coagulation Disorders, edited by Biggs and MacFarlane, published by F. A. Davis Co., Philadelphia, pp. 6269 (1966).

(d) Total protein content: Total protein content is determined by ultraviolet absorption at a wavelength of 280 my.

By the above assay procedures, the prothrombin complex of this example was found to be free of thrombin activity and to contain (on the average of many lots) about 8 mg. of protein per ml. The average Factor IX activity of these lots was about 20 to 40 times that of an equivalent volume of normal whole plasma; the aver age Factor II activity of these lots was about to times that of an equivalent volume of normal whole plasma. Since normal whole plasma contains about 70 mg. of protein per ml., the Factor IX activity in the prothrombin complex of this example is contained in only about one two-hundredth to one four-hundredth to the amount of protein present in plasma providing an equal amount of Factor IX activity and the Factor II activity is contained in only one fiftieth to one hundred fiftieth to the amount of protein present in plasma providing an equal amount of Factor II activity.

The prothrombin complex of the above example also contains high activity levels of Factors VII and X relative to the levels found in normal whole plasma. These factors are usually measured together in a determination of the proconvertin-Stuart-Prower complex.

EXAMPLE 4 The AHF-containing precipitate from Example 1 is resuspended in citrated saline solution to a volume equal to one-twentieth the volume of the original plasma which the precipitate represents. The resulting suspension (pH 6-7) is mixed with PEG 4000 to a concentration of 3.5% PEG. The mixture is gently agitated at room temperature for ten minutes, and then centrifuged for fifteen minutes at 5000 r.p.m. The supernate is decanted and adjusted to pH 6.88 with 0.1 normal sodium hydroxide. Additional polyethylene glycol 4000 is added to the solution to make the final PEG. concentration 10 percent. The

mixture is gently agitated at room temperature for thirty minutes, and centrifuged at 5000 rpm. for one-half hour.

The supernate is decanted and the precipitate is Washed in cold water (2 0.). Spin washing is then carried out for five minutes at 5000 r.p.m. at a temperature of 4 C. The supernate is decanted and the precipitate is redissolved in glycine citrated saline.

The redissolved precipitate is then adjusted to pH 6.88 with 0.1 normal acetic acid. By suitable means (for example, refrigeration or use of isopropanol Dry Ice bath) the solution is cooled to a temperature of from 6 C. to 10 C. To the cooled solution, sufficient glycine is added to make the solution 1.8 molal with respect to glycine. The mixture is gently agitated for 45 to 60 minutes at a temperature of from 2 C. to 10 C., and then centrifuged by continuous flow or bucket centrifugation. The resulting glycine precipitate is collected and gently washed with buffered water at a temperature of 0 C. to 4 C.

When the glycine precipitate has dissolved, it is preferable to clarify the solution by centrifugation and/or filtration using a 293 mm. Millipore filter (membranes used: 1.2 microns, 0.45 micron, and 0.3 micron).

The liquid human blood plasma product prepared in the above manner by the successive fractionation of the AHF-containing precipitate from Example 1, first by polyethylene glycol precipitation and then by glycine precipitation, has an AHF concentration of high potency. This liquid product is then frozen by shell freezing (60 C.) and storing in a flash freezer (20 C. to 30 C.) for at least three hours. The frozen product can then be retained under ordinary refrigeration conditions (4 0., preferably at 20 C. to -30 C.) without loss of its AHF activity for periods of time of one year and longer. This product when reconstituted can be administered intravenously to hemophiliacs as required by conventional transfusion means.

The AHF concentrate prepared by the procedure of this example contains less than 0. 05% (generally as little as 0.01%) residual polyethylene glycol and is highly soluble in water.

EXAMPLE 5 Example 4 is repeated including the additional step of purification of the AHF fraction with ECTEOLA cellulose resin in the following manner:

REAGENTS ECTEOLA cellulose resin (1) Mix 60 grams NaOH with 150 ml. H 0.

(2) Allow the mixture to cool.

(3) Place 60 grams cellulose (Whatman Cellulose Powder CF 11) in a beaker and mix thoroughly with the above NaOH solution.

(4) Allow the mixture to stand overnight (12 hours).

(5) On the next day prepare a solution of 35 ml. triethanolamine and 60 ml. of epichlorohydrin. Mix well under a hood.

(6) Add this solution quickly to the above cellulose and mix well. Place the reaction vessel out of a draft. (The reaction is exothermic and will heat to about C. The mixture will turn brown in one to two hours.)

(7) Cool the mixture at room temperature under a hood.

(8) Add 350 ml. 2 M NaCl in small portions.

(9i)i1 Filter this mixture through a coarse sintered glass ter.

(10) Wash the precipitate twice with 500 ml. of 1 N NaOH. (This removes deep discoloration.)

( l1) Suspend the precipitate in 350 ml. 1 N HCl in filter funnel. Apply a vacuum.

( 12) Repeat step 11 with 250ml. 1 N NaOH.

(13) Repeat step 11 with 250 ml. 1 N HCl.

( 14) Repeat step 11 with 250 ml. 1 N NaOH.

(15) Transfer the precipitate to a 3 liter beaker.

(16) Add 250 ml. 1 N NaOH. Mix.

(17) Add distilled water to the mixture to fill beaker;

mix. Cover and allow to stand overnight.

(18) Decant the supernatant.

(19) Add water to the precipitate to fill beaker and mix.

(20) Wash the mixture on the filter with water. (Four liters or more, until a negative test for alkali with 1% alcoholic phenolphthalein is obtained.)

(21) Make a final wash with two 250 ml. portions absolute ethanol.

(22) Place on filter paper. Mash and spread and place it to dry overnight.

Chloride buffer:

0.8% NaCl (8 gm./l.) I 0.02 M imidazole (1.36 gm./l.) Adjust pH to 6.9 with 1 N HCl Eluting buffer:

0.5 M NaCl 0.02 M imidazole (1.36 gm./l.) Adjust pH to 6.9 with 1 N HCl A commercially available ECTEOLA cellulose resin which is first recycled with NaOH, for example, as in the following manner, can be used in place of the above-prepared ECTEOLA cellulose resin.

Recycled commercial ECTEOLA cellulose resin (1) Mix 60 grams commercial ECTEOLA cellulose resin with 350 ml. 2 M NaCl.

(2) Filter this mixture through a coarse sintered glass filter.

(3) Wash the precipitate two times with 500* ml. 1 N

NaOH.

(4) Wash one time with 350 ml. 1 N HCl.

(5) Wash one time with 250 ml. 1 N NaOH.

(6) Wash one time with 250 ml. 1 N HCl.

(7) Wash one time with 250 ml. 1 N NaOH.

(8) Transfer the precipitate to a 3 liter beaker and add 250 ml. 1 N NaOH. Mix.

(9) Add distilled water to the mixture to fill beaker. Mix.

Cover and let stand overnight.

(10) Decant the liquid; add 3 liters water to the precipitate; mix. Filter.

(11) Wash the precipitate with water until phenolphthalein test is negative.

(12) Wash the precipitate two times with 500 ml. ab-

solute .ethanol.

(13) Air-dry the precipitate on filter paper.

PROCEDURE When the starting material for the purification with ECTEOLA cellulose resin is a polyethylene glycol-precipitated fraction of AHF, the AHF fraction is first dissolved in chloride buffer. When the starting material is a glycineprecipitated fraction of AHF, the AHF fraction is first dialyzed against the chloride butfer for one hour to remove glycine and reduce its ionic strength. Purification of the buffered AHF fraction by column technique with the above-prepared ECTEOLA cellulose resin proceeds as follows:

The ECTEOLA cellulose resin is equilibrated overnight (12 hours) in a 5 C. box by mixture with chloride bulfer in proportions of 15 grams of resin to 600 ml. buffer. The resulting resin slurry is poured on a column of from one to one and one-half (1 /2) inches in diameter x 18 inches high. After the buffer comes down to the level of the resin, the AHF fraction is adjusted to two and one-half (2%) ml. per minute and the amount of AHF fraction applied to a single column is from 1000 to 3500 units of AHF. (one unit of AHF is equal to the AHF activity in one cc. of normal human blood plasma.) After the AHF has been applied to the column, from 200 to 500 ml. chloride buffer is washed through the resin. When the chloride buffer comes down to the level of the resin, the eluting buffer is applied to the column. The eluate is collected in ten ml. portions and analyzed for protein, fibrinogen and AHF activity.

The eluate portions having the most active AHF activity are retained and stabilized by the addition of 1% albumin. The stabilized solution is then filtered using a 293 mm. Millipore filter as in Example 4. A silver filter of the same size can also be used in place of the Millipore filter. This final liquid product can then be frozen by shell freezing, followed by storage in a flash freezer for at least three hours, and then retained under ordinary refrigeration conditions in the manner of the final product of Example 4.

Human whole blood plasma, and bovine and porcine plasma and AHF-containing plasma fractions can be fractionated by the successive polyethylene glycol and glycine precipitation procedures described herein to produce AHF concentrates similar to those described above in Examples 4 and 5.

Various modifications and adaptations of the present invention can be devised, after reading the foregoing specification and the claims appended hereto, by the person skilled in the art without departing from the spirit and scope of the invention. All such variations and modifications are included Within the scope of the invention as delined in the following claims.

What is claimed is:

1. A method for the preparation of a prothrombin complex from citrated blood plasma comprising fractionating citrated blood plasma by mixing with glycine having a molarity of from about 1.3 to about 1.8 to produce an AHF-containing precipitate and a prothrombin complex-containing supernatant, fractionating said prothrombin complex-containing supernatant by mixing with polyethylene glycol having a molecular weight of from about 200 to about 20,000 to a concentration of about 30% by weight and then separating the resulting prothrombin complex-containing precipitate from the undesired citrate and glycine salt-containing supernatant, suspending said prothrombin complex-containing precipitate in normal physiological saline, adjusting the suspension to a pH of from about 6.8 to about 7.2, thoroughly mixing the supernatant with tribasic calcium phosphate to adsorb the coagulation factors, thoroughly mixing the tricalcium phosphate adsorbed protein precipitate with from about 0.05 M to about 0.2 M trisodium citrate followed by recovery of the resulting supernatant, and then subjecting the recovered supernatant to a succession of two precipitations with polyethylene glycol having a molecular weight of from about 200 to about 20,000, first at a pH of from about 6.8 to about 8.0 and a final concentration of from about 5% to about 10% polyethylene glycol with retention of the resulting supernatant, and then at a pH of from about 5.0 to about 5.4 and a final concentration of at least about 20% polyethylene glycol by Weight of said retained supernataint with retention of the resulting precipitate as the active prothrombin complex.

2. The method of claim 1 in which the polyethylene glycol has an average molecular weight of from about 400 to about 6,000.

3. The method of claim 1 in which the polyethylene glycol has an average molecular weight of about 4,000.

4. A method for the preparation of a concentrate of AHF from citrated blood plasma comprising fractionating citrated blood plasma by mixing with glycine having a molarity of from about 1.3 to about 1.8 to produce an AHF-containing precipitate and a prothrombin complexcontaining supernatant, fractionating said AHF-containing precipitate by mixing with polyethylene lycol having a molecular weight of from about 200 to about 20,000 to a concentration of from about 3% to about 4% by weight, mixing the AHF-containing supernatant with said polyethylene glycol to a concentration of about 10% by Weight, mixing the resulting AHF-containing precipitate with glycine having a molarity of about 1.8 and recoverlIlg the remaining precipitate as the active concentrate of AHF.

5. The method of claim 1 in which the polyethylene glycol has an average molecular weight of from about 400 to about 6,000.

8,682,881 1 1 12 6. The method of claim 1 in which the polyethylene OTHER REFERENCES glycol has an average molecular weight of about 4,000. A I n 1 f th M L 5 7. The method of claim 1 including the additional step 1965m 3322 2 3 z z lcal Sciences v0 2 of purification with triethanolamino-ethylated cellulose jollmal of Hemflflggy (Blood), vol. 28, p. 1011, Dec. Tesm- 5 5, 1966, Johnson et al.

References Cited HOWARD E. SCHAIN, Primary Examiner UNITED STATES PATENTS 3,415,804 12/1968 P015011 260-112 US. Cl. XRI

Fekete et a1. 

